Genomic Analysis of Two Representative Strains of Shewanella putrefaciens Isolated from Bigeye Tuna: Biofilm and Spoilage-Associated Behavior

Shewanella putrefaciens can cause the spoilage of seafood and shorten its shelf life. In this study, both strains of S. putrefaciens (YZ08 and YZ-J) isolated from spoiled bigeye tuna were subjected to in-depth phenotypic and genotypic characterization to better understand their roles in seafood spoilage. The complete genome sequences of strains YZ08 and YZ-J were reported. Unique genes of the two S. putrefaciens strains were identified by pan-genomic analysis. In vitro experiments revealed that YZ08 and YZ-J could adapt to various environmental stresses, including cold-shock temperature, pH, NaCl, and nutrient stresses. YZ08 was better at adapting to NaCl stress, and its genome possessed more NaCl stress-related genes compared with the YZ-J strain. YZ-J was a higher biofilm and exopolysaccharide producer than YZ08 at 4 and 30 °C, while YZ08 showed greater motility and enhanced capacity for biogenic amine metabolism, trimethylamine metabolism, and sulfur metabolism compared with YZ-J at both temperatures. That YZ08 produced low biofilm and exopolysaccharide contents and displayed high motility may be associated with the presence of more a greater number of genes encoding chemotaxis-related proteins (cheX) and low expression of the bpfA operon. This study provided novel molecular targets for the development of new antiseptic antisepsis strategies.


Introduction
Aquatic products are regarded as an important food source globally owing to its low-fat content and rich animal protein. However, aquatic products are highly perishable foods after death, even under refrigerated conditions. Microorganisms are essential in the spoilage of aquatic products, even with the rapid development of modern preservation technologies [1,2]. The main microorganisms responsible for food spoilage are known as specific spoilage organisms (SSOs) [3]. These SSOs can break down nitrogenous compounds (amino acids and proteins) in aquatic products into ammonia, biogenic amines, sulfides, and volatile compounds (including aldehydes, ketones, alcohols, and organic acids), leading to the degradation of sensory properties and making fish products unacceptable [4,5].
Although low temperatures inhibit the growth and metabolism of most microbes, many studies showed that S. putrefaciens can reduce the shelf life of refrigerated seafood, such as tuna [6], Pacific white shrimp [7], and cod [8]. Most S. putrefaciens strains can reduce trimethylamine oxide (TMAO) to trimethylamine (TMA) [9] and decarboxylate specific amino acids to biogenic amines, including putrescine, histamine, and cadaverine [10]. sequencing library construction. Two libraries with insert sizes of approximately 400 bp and 15-20 kb was constructed and sequenced on an Illumina NovaSeq 6000 sequencing platform and a PacBio RSII platform, respectively. Raw data obtained from the Illumina NovaSeq 6000 sequencing platform and PacBio RSII platform were quality controlled (remove lowquality reads and repeat reads) for further assembly. First, Illumina sequencing data were assembled using SOAPdenova v1.05 and then were compared with the PacBio sequencing data corrected errors. Next, the corrected data were assembled using Celera Assembler v8.0 to generate the scaffolds. Finally, the assembled scaffolds were mapped back to Illumina clean reads using GapCloser v1.12 for gap closing.
For the identification of genes associated with spoilage behavior, key proteins related to spoilage metabolism were collected. Candidate genes were obtained by searching all predicted proteins in the YZ08 and YZ-J genomes (E-value = 1E10; coverage ≥ 70%; identity ≥ 35%) using the blastP algorithm. Candidate genes were further confirmed by protein, COG, and GO functional annotation [18]. In addition, relevant metabolic genes were obtained directly by KEGG pathway analysis.

Pan-Genome Analysis and Genome Mining of S. putrefaciens YZ08 and YZ-J
Pairwise genome alignment and visualization analysis of S. ptrefaciens YZ08 and YZ-J strains were performed using MAUVE v2. 4.0 (University of Wisconsin, Wisconsin, USA) [19]. All 10 S. putrefaciens complete genomes in NCBI were subjected to pan/core genome analysis using the Bacterial Pan Genome Analysis tool (BPGA v1.3) [20]. The NCBI accession numbers for 8 of the 10 strains (excluding strains YZ08 and YZ-J) were NZ_CP066370, NZ_CP066369, NZ_CP046329, LR134303, CP015194, NZ_CP078038, NZ_CP028435, and CP070865. Each nucleotide sequence was analyzed using default settings. In BPGA, homologous protein clusters were identified using USEARCH (a clustering tool) with a threshold of 0.5 and a phylogenetic tree was constructed based on the core genome. In addition, pan-genomic analysis was performed for strains YZ08 and YZ-J and unique genes were subjected to COG and KEGG functional annotation.

Growth and Biofilm Formation of S. putrefaciens YZ08 and YZ-J under Stress Conditions
S. putrefaciens strains YZ08 and YZ-J were pre-cultured in Luria Broth (LB) medium at 30 • C for 12 h. As a control, each strain was inoculated into LB medium (pH 7.0) at a ratio of 1:1000 and incubated at 30 • C. To induce pH stress, the incubation temperature was kept constant (30 • C), but the pH of the LB medium was regulated to 6.0 using HCl; for NaCl stress, the NaCl concentration was adjusted to 5% by adding NaCl; for nutrient stress, the medium was diluted to 20% by adding distilled water; for temperature stress, the temperature was set at 4 • C. The bacteria were incubated under stress conditions for 168 h, and the total cell numbers was measured every 12 h. The total cell numbers were performed using plate count agar (PCA). Samples (1 mL) were serially diluted 10-fold with normal saline, and dilutions (0.1 mL) were spread on PCA. Plates were placed at 30 • C for 48 h and the total cell numbers were determined.
One milliliter of the dilutions (1:1000) of the above pre-cultures under various stress conditions were transferred to a 48-well plate. The plates were incubated under static conditions for 12, 24, 48, and 72 h. Biofilm formation were determinate by Yan and Xie [11]. Briefly, after culture, the supernatant was carefully discarded and the adherent cells were washed twice using saline (0.85%) and dried, then stained with 0.2% crystal violet for 15 min, washed and dried. Finally, it was dissolved in 95% ethanol to determine OD 600 . The total cell numbers in LB medium were also determined for normalization. The total cell numbers were determined by serial dilution as described in Section 2.4. The normalized results were expressed as the ratio of the OD 600 to the total number of cells.

Motility Assays
Swarming and swimming motility were measured on LB cultures containing 1.5% and 0.3% agar, respectively. Five microliters of bacterial culture (1 × 10 8 CFU/mL) was dropped onto agar plates and incubated at 4 • C and 30 • C for 24, 48, and 72 h, and the diameters of the motility zones were measured, respectively.

Proteolytic and Lipolytic Activity
Proteolytic activity was measured on agar plates containing 5% skimmed milk (prepared with deionized water containing 5% skimmed milk powder and 2% agar), and lipolytic activity was measured on triglyceride agar (Solarbio, Beijing, China). Incubation was performed at 30 • C for 24, 48, and 72 h and 4 • C for 1, 2, and 4 days, respectively. Finally, proteolytic and lipolytic activity was determined according to the size of the produced halos.

Determination of TMA Content
Both strains of S. putrefaciens were inoculated in LB medium with TMAO (10 mM) and phosphate buffer saline (PBS, 100 mM) for 6 days at 4 • C and 48 h at 30 • C. TMA content was determined using the picric acid method [21].

Preparation and Inoculation of Sterile Tuna Juice
Minced bigeye tuna back muscle (2 kg) was homogenized and boiled for 5 min with 2 L of distilled water. After adding 1.6 g/L TMAO, 40 mg/L L-cysteine and Lmethionine, the filtrate was sterilized (121 • C, 15 min), yielding sterile bigeye tuna juice. The S. putrefaciens strains were separately inoculated into the fish juice at a final concentration of approximately 5 log CFU/mL and stored at 4 and 30 • C.

Determination of H 2 S Content
The H 2 S content in fish juice was determined using a H 2 S concentration determination kit (Beijing Solabao, Beijing, China) and was expressed as µmol/mL.

Determination of Exopolysaccharide Content and Extracellular Protease Activity
The exopolysaccharide of the inoculated fish juice was extracted according to the method of Feng et al. [24]. Nine milliliters of the inoculated fish juice was transferred to a six-well plate and incubated at 30 • C for 24, 48, and 72 h and at 4 • C for 1, 2, and 4 days, respectively. After carefully removing the cultures from the wells, the plate was cleaned 3 times with PBS to remove residual bacterial cells, 9 mL of PBS was added to each well, followed by sonication at 50 kHz for 5 min to dissolve the exopolysaccharides that had adhered to the walls of the wells (sonication can lyse cells in biofilms, resulting in the release of intracellular content). After centrifugation at 12,000× g for 15 min at 4 • C, the obtained supernatant was used to measure exopolysaccharide content using the phenol-sulfuric acid method [25], with glucose serving as a standard. Exopolysaccharide content was expressed as µg/mL. Extracellular protease activity was determined according to Anbu [26], with some modifications. After centrifugation at 10,000× g at 4 • C, the supernatant (500 µL) of the fish juice was added to an equal volume of 1% (w/v) casein substrate solution and incubated at 37 • C for 10 min. The reaction was terminated by adding 1 mL of TCA (20%). Finally, after centrifugation (12,000× g, 10 min), the tyrosine content was determined using the Folin method [27]. The results were expressed as U/mL.

RT-qPCR
S. putrefaciens strains YZ08 and YZ-J were inoculated in LB medium at 30 • C until the log phase (OD 600 = 0.8). Total RNA was isolated from cultured cells using a Spin Column Bacteria Total RNA Purification Kit (Sangon, Shanghai, China) and reverse-transcribed into cDNA using an AMV First Strand cDNA Synthesis Kit (Sangon, Shanghai, China) with random primer p(dN) 6 . qPCR was performed using SG Fast qPCR Master Mix (SYBR Green) (Sangon, Shanghai, China). The sequences of the primers used for qPCR are listed in Table S1. mRNA levels were normalized to that of the 16 S rRNA gene. The relative expression levels of each gene were determined using the 2 −∆∆Ct method [28].

Statistical Analysis
All data were analyzed using SPSS 19.0 (IBM, Chicago, IL, USA). The Student's t-test was employed for comparisons between two groups. One-way analysis of variance (ANOVA) with Duncan's post-hoc test was used for multiple groups. p < 0.05 were considered significant. All experiments were repeated at least three times.

Identification and Genome Properties of S. putrefaciens Strains YZ08 and YZ-J
The complete genome sequences were submitted in GenBank with accession numbers CP080633 (for YZ08) and CP080635 (for YZ-J). The genomes of S. putrefaciens YZ08 and YZ-J were determined to be 5,019,740 bp and 4,386,160 bp long, with average GC contents of 47.69% and 46.53%, respectively (

COG, GO, and KEGG Function Classification Analysis of the Two Strains
The unknown function category contained the largest number of genes for both S. putrefaciens strains. Both strains showed similar trends by which amino acid transport and metabolism, and energy production and conversion were the main COGs ( Figure 2A). For energy production and conversion, the four most abundant COGs were COG0243, COG1012, COG0437, and COG1301 for YZ08, and COG1012, COG1053, COG0243, and COG0437 for YZ-J. COG0437 plays a crucial role in various electron transfer processes and several enzymatic reactions [29]. In response to environmental stresses, two Na+ ions were transported by COG1301 [30]. A similar COG functional classification was also found in S. baltica 128 (accession number CP028730). The differences in COG classification may be indicative of differences in metabolism and adaptability to the environment between the two strains. S. putrefaciens YZ08 and YZ-J genes were assigned to three function classifications by GO analysis ( Figure 2B). The genes of the YZ08 and YZ-J strains in the biological process category were divided into 20 subfunctions, most of which were associated with cellular processes (GO:0009987), metabolic processes (GO:0008152), and response to stimulus (GO:0050896), which was consistent with the features reported for Shewanella spp. [13]. A similar GO functional classification was found for S. putrefaciens XY07 (accession number CP070865). The results for the KEGG pathway analysis of S. putrefaciens YZ08 and YZ-J genes are shown in Figure 2C,D. In the five KEGG pathways of the two strains, metabolism comprised the largest number of genes, followed by environmental information processing, cellular processes, and genetic information processing. Some pathways may be related to the distinct phenotypes shown by the two strains, such as two-component system (ko02020), microbial metabolism in diverse environments (ko01120), biosynthesis of amino acids (ko01230), bacterial chemotaxis (ko02030), ABC transporters (ko02010), biofilm formation-Vibrio cholerae (ko05111), flagellar assembly (ko02040), and cysteine and methionine metabolism (ko00270). Two-component systems (TCS) can translate extracellular signals into gene expression patterns that facilitate bacterial regulation of various physiological functions. ABC transporters (ko02010) are transmembrane proteins that use energy to transport substrates into the cell [31].

Genome Synteny and Pan-Genome Analysis
To explore the genetic differences between both strains, a genome synteny and pangenome analysis were also investigated. As shown in Figure 3A, analysis of the genome sequences using MAUVE revealed that S. putrefaciensYZ08 and YZ-J shared many homologous regions. Although large genomic rearrangements and inversions were found in both strains, overall, the YZ08 and YZ-J genomes shared a large number of homologous regions.
The pan-genome of the 10 S. putrefaciens strains included 2637 core genes, 10,413 accessory genes and 1944 unique genes. Analysis of the pan-genome and core genome maps of oral S. putrefaciens ( Figure 3B,C) showed that the number of the pan-genome increased, while that of the core genome decreased, indicating that S. putrefaciens has an open pangenome and has the ability to survive in a variety of environments [13]. To investigate the phylogenetic relationships among the 10 strains, a phylogenetic tree was generated based on 2637 core genes ( Figure 3D). S. putrefaciens YZ08 showed the greatest genetic relationship with the pap11 strain, while YZ-J was close to XY07, ATCC8071, and WS13.

Genome Synteny and Pan-Genome Analysis
To explore the genetic differences between both strains, a genome synteny and pangenome analysis were also investigated. As shown in Figure 3A, analysis of the genome sequences using MAUVE revealed that S. putrefaciensYZ08 and YZ-J shared many homologous regions. Although large genomic rearrangements and inversions were found in both strains, overall, the YZ08 and YZ-J genomes shared a large number of homologous regions. The pan-genome of S. putrefaciens YZ08 and YZ-J was investigated. The unique genes of both strains were listed in Tables S2 and S3. The unique genes of the two strains were annotated using KEGG and COG distribution ( Figure 3E,F). In COG distribution, many unique genes of both strains were related to signal transduction mechanisms, which were involved in the regulation of various life activities of microorganisms. Key biofilm regulatory genes were identified in strain YZ08, annotated as COG5001 (diguanylate cyclase phosphodiesterase) in COG and classified as ko05111 (biofilm formation-Vibrio cholerae) in KEGG. These genes may play an inhibitory role in biofilm formation [32]. YZ08 may possess stronger amino acid metabolic activity than YZ-J owing to a greater abundance of COG0834 components (ABC-type amino acid transport/signal transduction system). KEGG distribution showed that only YZ08 was involved in trimethylamine metabolism in methane metabolism (ko00680). Both strains contained genes coding for proteins with functions in membrane transport (ABC transporters). These transporters are involved in transporting a large number of endogenous substrates and exogenous compounds across lipid membranes and are associated with many important biological processes, such as the release of secreted proteins, cellular detoxification, lipid homeostasis, ion channel regulation, and ribosome assembly [33]. In addition, COG and KEGG distributions indicated that YZ08 possessed more cell motility-related genes than YZ-J, especially those associated with bacterial chemotaxis. In conclusion, the pan-genomic analysis provided new insights into the differential genetic content of the two strains.  The pan-genome of the 10 S. putrefaciens strains included 2637 core genes, 10,413 accessory genes and 1944 unique genes. Analysis of the pan-genome and core genome maps of oral S. putrefaciens ( Figure 3B,C) showed that the number of the pan-genome increased, while that of the core genome decreased, indicating that S. putrefaciens has an open pangenome and has the ability to survive in a variety of environments [13]. To investigate the phylogenetic relationships among the 10 strains, a phylogenetic tree was generated based on 2637 core genes ( Figure 3D). S. putrefaciens YZ08 showed the greatest genetic relationship with the pap11 strain, while YZ-J was close to XY07, ATCC8071, and WS13.

Stress Adaptation
During food processing, microbes are subjected to a range of stresses, such as temperature, salt, pH, and nutrition stresses. The growth and biofilm formation of S. putrefaciens YZ08 and YZ-J under different stress conditions are shown in Figure 4. The growth of both YZ08 and YZ-J in 5% NaCl and 20% LB was reduced compared with that in the control group ( Figure 4A). The lag period for the growth of the two strains in the 4 • C groups was substantially longer than that of the control, but the maximum cell concentrations of the two strains were similar, which was consistent with the previous study [15]. The growth rate of YZ08 was higher than that of YZ-J in 5% NaCl; however, no significant differences were observed between the two strains for the other stress conditions. The Normalized biofilm formation rates of two strains in the 5% NaCl, 20% LB and 4 • C groups were higher than those in the control group, reaching significance in the 4 • C groups for both strains (p < 0.05) ( Figure 4B). Low temperatures promote the expression of related genes to enhance the formation of biofilms [11]. Similarly, the higher normalized biofilm formation rates recorded under pH 6.0, 5% NaCl, and 20% LB stress relative to the control condition may be related to the formation of a greater amount of biofilm to protect the bacteria under stressful conditions [13]. In addition, the normalized biofilm decreased from 24 h to 144 h in the control, pH 6.0, NaCl 5%, and 4 • C groups, likely because the biofilm at this stage was in the dispersal period [34]. In all groups, the normalized biofilm formation of YZ08 was lower than that of YZ-J.
provided new insights into the differential genetic content of the two strains.

Stress Adaptation
During food processing, microbes are subjected to a range of stresses, such as temperature, salt, pH, and nutrition stresses. The growth and biofilm formation of S. putrefaciens YZ08 and YZ-J under different stress conditions are shown in Figure 4. The growth of both YZ08 and YZ-J in 5% NaCl and 20% LB was reduced compared with that in the control group ( Figure 4A). The lag period for the growth of the two strains in the 4 °C groups was substantially longer than that of the control, but the maximum cell concentrations of the two strains were similar, which was consistent with the previous study [15]. The growth rate of YZ08 was higher than that of YZ-J in 5% NaCl; however, no significant differences were observed between the two strains for the other stress conditions. The Normalized biofilm formation rates of two strains in the 5% NaCl, 20% LB and 4 °C groups were higher than those in the control group, reaching significance in the 4 °C groups for both strains (p < 0.05) ( Figure 4B). Low temperatures promote the expression of related genes to enhance the formation of biofilms [11]. Similarly, the higher normalized biofilm formation rates recorded under pH 6.0, 5% NaCl, and 20% LB stress relative to the control condition may be related to the formation of a greater amount of biofilm to protect the bacteria under stressful conditions [13]. In addition, the normalized biofilm decreased from 24 h to 144 h in the control, pH 6.0, NaCl 5%, and 4 °C groups, likely because the biofilm at this stage was in the dispersal period [34]. In all groups, the normalized biofilm formation of YZ08 was lower than that of YZ-J. A series of stress-related genes of S. putrefaciens YZ08 and YZ-J, including temperature, pH, NaCl, and nutrient stresses, is shown in Table 1. The cold shock genes cspA and A series of stress-related genes of S. putrefaciens YZ08 and YZ-J, including temperature, pH, NaCl, and nutrient stresses, is shown in Table 1. The cold shock genes cspA and cspD in L. monocytogenes are required to induce its growth at low temperatures [35,36] and may exert a similar function in S. putrefaciens YZ08 and YZ-J. Three cspA/cspD genes (K2227_07410, K2227_08825, K2227_12570, and K3G22_06460, K3G22_07485, K3G22_10845, respectively) were identified in the YZ08 and YZ-J genomes, which may explain the similar cold adaptability of the two strains. Furthermore, the genomes of both YZ08 and YZ-J contained eight genes encoding stress-related F0F1 ATP synthase, which is associated with the synthesis of ATP using ion translocation [37]. Interestingly, YZ08 contained six genes encoding sodium: proton antiporter and one encoding a transporter protein (K2227_13575) related to osmotic pressure, whereas YZ-J contained only four genes encoding sodium: proton transporters and none coding for the osmotic pressure-related transporter protein. This observation may partially explain the better growth of the YZ08 strain under 5% NaCl relative to that of strain YZ-J. The presence of genes encoding choline/glycine/proline betaine transporter and plasma membrane protein involved in salt tolerance indicated that S. putrefaciens maintains osmotic balance using a compatible solutes strategy when exposed to osmotic stress. RT-qPCR was used to study the expression of osmotic stressrelated genes (encoding choline/glycine/proline betaine transporter and plasma membrane protein-K2227_07790 and K2227_01670, respectively, in YZ08 and K3G22_06825 and K3G22_01330, respectively, in YZ-J). As shown in Figure 5, the expression of the genes encoding choline/glycine/proline betaine transporter were significantly higher in strain YZ08 than in strain YZ-J (p < 0.001), which was consistent with YZ08 being better adapted to a high salt environment relative to YZ-J. However, the expression levels of the gene encoding the plasma membrane protein did not show significant differences between in the two strains (p > 0.05). In addition, YZ08 and YZ-J shared similar genes encoding amino acid synthases.

Motility
Cell surface characteristics (chemotactic systems) and motility are critical during biofilm formation [38]. S. putrefaciens motility (swimming and swarming) is shown in Figure  6. The swimming motility of both YZ08 and YZ-J increased in a time-dependent manner at the optimal growth temperature (30 °C ), but swimming behavior began late at low temperatures (4 °C ), especially for strain YZ-J ( Figure 6A). The swimming ability of YZ08 strain at the two temperatures was stronger than that of strain YZ-J, which may have been due to the stronger movement ability of the polar flagella of YZ08, as previously described for Vibrio parahaemolyticus RIMD2210633. [39]. The swimming abilities of the two S. putrefaciens strains were stronger than those of S. baltica SB02 and S. baltica OS155 [24,40]. Differences in genes encoding chemotaxis proteins and the regulation of some differential

Motility
Cell surface characteristics (chemotactic systems) and motility are critical during biofilm formation [38]. S. putrefaciens motility (swimming and swarming) is shown in Figure 6. The swimming motility of both YZ08 and YZ-J increased in a time-dependent manner at the optimal growth temperature (30 • C), but swimming behavior began late at low temperatures (4 • C), especially for strain YZ-J ( Figure 6A). The swimming ability of YZ08 strain at the two temperatures was stronger than that of strain YZ-J, which may have been due to the stronger movement ability of the polar flagella of YZ08, as previously described for Vibrio parahaemolyticus RIMD2210633. [39]. The swimming abilities of the two S. putrefaciens strains were stronger than those of S. baltica SB02 and S. baltica OS155 [24,40]. Differences in genes encoding chemotaxis proteins and the regulation of some differential key genes such as flgM, encoding an important regulatory factor for flagella gene expression; zomB, encoding a flagellar motor control protein; and genes encoding PilZ domain proteins, may explain the different swimming phenotypes of the two strains [41,42]. In contrast to their strong swimming abilities, the swarming abilities of S. putrefaciens YZ08 and YZ-J were weak, and also showed a time dependence (Figure 6B). At 30 • C, the difference in swarming ability between the two strains was not significant, while at 4 • C the swarming ability of YZ08 was slightly stronger than that of YZ-J. The absence of lateral flagella may explain the weaker swarming ability of Shewanella spp. relative to Vibrio spp. [39,43]. No genes encoding lateral flagella were found in either strain in this study, which would account for their weak swarming ability.   Table 2 lists most of the motility-associated genes in YZ08 and YZ-J. No differences were found between the two strains for the three gene clusters encoding polar flagellins (A-I, A-II, and A-III). A-I contains structural genes coding for sodium-driven motor rings, loops and hook proteins, and assembly and chaperone proteins, as previously described in Vibrio [44]. The A-II gene cluster contains genes encoding regulatory proteins, filaments, basal bodies, switch proteins, and export proteins. The deletion of these genes can lead to the loss of motility in bacteria, as previously described in Pseudomonas fluorescens F113 [45]. The third cluster (A-III) contains chemotaxis genes, export genes, and regulatory genes that express late flagellar genes encoding filament proteins, motor proteins, and other flagellar-associated secretory proteins, as previously described [46]. In addition to genes encoding flagellins, those coding for chemotaxis proteins are also critical for bacterial motility [47]. The YZ08 strain contained up to 19 genes encoding chemotaxis proteins compared with only 13 for YZ-J (Table 2), which may explain the markedly greater swimming ability of YZ08 relative to that of YZ-J.   Table 2 lists most of the motility-associated genes in YZ08 and YZ-J. No differences were found between the two strains for the three gene clusters encoding polar flagellins (A-I, A-II, and A-III). A-I contains structural genes coding for sodium-driven motor rings, loops and hook proteins, and assembly and chaperone proteins, as previously described in Vibrio [44]. The A-II gene cluster contains genes encoding regulatory proteins, filaments, basal bodies, switch proteins, and export proteins. The deletion of these genes can lead to the loss of motility in bacteria, as previously described in Pseudomonas fluorescens F113 [45]. The third cluster (A-III) contains chemotaxis genes, export genes, and regulatory genes that express late flagellar genes encoding filament proteins, motor proteins, and other flagellarassociated secretory proteins, as previously described [46]. In addition to genes encoding flagellins, those coding for chemotaxis proteins are also critical for bacterial motility [47]. The YZ08 strain contained up to 19 genes encoding chemotaxis proteins compared with only 13 for YZ-J (Table 2), which may explain the markedly greater swimming ability of YZ08 relative to that of YZ-J.

Spoilage-Related Metabolic Pathways
S. putrefaciens usually generates spoilage metabolites such as total volatile base nitrogen (TVB-N), TMA, and biogenic amines (BAs) in seafood, leading to a decline in its quality [7]. The spoilage potential of S. putrefaciens is associated with sulfur metabolism, BAs metabolism, TMA metabolism, and protease secretion [2].

Biogenic Amines (BAs) Metabolism
Putrescine, cadaverine, and histamine are common BAs found in spoiled tuna [48]. However, as S. putrefaciens is mainly associated with the production of putrescine and cadaverine, and generates only limited amounts of histamine [49]. We focused on investigating the putrescine and cadaverine production activities in the two S. putrefaciens strains. The amounts of putrescine and cadaverine produced by YZ08 and YZ-J at 30 and 4 • C using ornithine, arginine, and lysine as substrates are shown in Figure 7A,B. The findings indicated that YZ08 produced greater amounts of putrescine and cadaverine than YZ-J at both the optimum growth temperature and low temperature. We also found that both S. putrefaciens strains produced more putrescine than cadaverine, which may be because putrescine can be produced using different substrates (ornithine and arginine) and through different pathways, whereas cadaverine is produced through only one pathway (lysine decarboxylation) [22].  The genomic analysis identified several BA-related genes in the two tested strains ( Table 2). Several pot genes involved in putrescine metabolism were identified in S. putrefaciens, including genes encoding substrate-binding proteins of the putrescine transport The genomic analysis identified several BA-related genes in the two tested strains (Table 2). Several pot genes involved in putrescine metabolism were identified in S. putrefaciens, including genes encoding substrate-binding proteins of the putrescine transport system, a putrescine transport ATP-binding protein, spermidine/putrescine ABC transporter permease, putrescine transport system permease protein, and a putrescine-ornithine antiporter. In addition, genes encoding putrescine importer PuuP and gamma-glutamylputrescine oxidoreductase were also found in the genomes of both strains. In S. putrefaciens CN32, ornithine decarboxylase is a key enzyme capable of producing putrescine from L-ornithine [2]. Similarly, arginine is converted to cadaverine by ornithine/arginine decarboxylase. The presence of ornithine/arginine decarboxylase corroborated the production of putrescine and cadaverine by the two strains. Although no difference was found in putrescine-related genes, the levels of putrescine and cadaverine production in YZ08 and YZ-J were different, indicating that some regulatory factors could induce the expression of these genes. The results of qRT-PCR supported this hypothesis, indicating that the expression of speC in S.putrefaciens YZ08 was significantly higher than that in YZ-J (p < 0.01, Figure 5).

TMA Metabolism
As mentioned earlier, most Shewanella spp. can reduce TMAO to TMA and produce a fishy odor. Figure 7C shows the amount of TMA produced by YZ08 and YZ-J in LB medium containing TMAO at 4 and 30 • C. At both temperatures, the amount of TMA produced by YZ08 was significantly higher than that of YZ-J (p < 0.05). As shown in Table 2, genes encoding trimethylamine N-oxide reductase system protein TorE (K2227_16520), pentaheme c-type cytochrome TorC (K2227_16525), trimethylamine-N-oxide reductase TorA (K2227_16530), molecular chaperone TorD (K2227_16535), histidine kinase TorS (K2227_16540), periplasmic protein TorT (K2227_16545), and response regulator TorR (K2227_16550) were found in YZ08, but not found in YZ-J. The same TMA metabolism related genes were identified in other strains (S. baltica OS155 and 128) [13,50]. Although no trimethylamine metabolism-related genes were found in YZ-J, this strain also produced small amounts of TMA, suggestive of the existence of other trimethylamine metabolism pathways. It has been reported that gut microbiota can metabolize compounds containing trimethylamine groups to produce TMA from the precursors of TMA containing choline, phosphatidylcholine, and glycerophosphatidylcholine. The key genes involved in this process are cutC, encoding a choline TMA-lyase and gene cutD, encoding a choline TMAlyase activase [51]. In the present study, pflA/D genes, homologs of cutC/D were found in S. putrefaciens YZ08 and YZ-J. cutD and pflD are related to pyruvate formate lyase activating enzyme, and cutC and pflA are homologous to pyruvate formate lyase. Therefore, a small amount of TMA produced in S. putrefaciens YZ-J may be related to the presence of pflA/D.

Sulfur Metabolism
H 2 S gas has a characteristic off-odor and is associated with the presence of Shewanella spp. during the spoilage of seafood [2]. In this study, we explored the H 2 S content produced by S. putrefaciens YZ08 and YZ-J ( Figure 7D). At 30 • C, YZ08 produced a significant amount of H 2 S in the fish juice. However, at the low temperature (4 • C), both strains generated low amounts of H 2 S at the end of storage (144 h). In general, YZ08 metabolized more H 2 S than YZ-J. The genes associated with sulfur metabolism in YZ08 and YZ-J are listed in Table 2. Sulfate is converted to adenosine 5 -phosphosulfate (APS) by sulfate adenylyltransferase (encoded by the cysN gene). APS is then converted to 3 -phosphonoadenosine-5 -phosphate sulfate (PAPS) by the action of adenylyl-sulfate kinase (encoded by cysC), which is then further reduced to sulfite by phosphonoadenosine phosphate reductase (encoded by cysH). Finally, sulfite is reduced to sulfide by dissimilatory sulfite reductase (encoded by sirA). Moreover, the ttrSRBC encoding tetrathionate response regulatory protein, tetrathionate sensor histidine kinase, tetrathionate reductase subunit B and cysteine synthase C was also identified in the genomes of both S. putrefaciens strains, suggesting that tetrathionate may be reduced and eventually form sulfide through the activity of these enzymes, consistent with the findings of Leustek et al. [52]. That the two strains contained the same sulfur metabolism genes, suggests that they produce different amounts of H 2 S. This could be explained by differences in the transcription levels given that the level of SirA was significantly greater in YZ08 than in YZ-J (p < 0.01) ( Figure 5). Highly similar genes related to sulfur metabolism were found in S. baltica 128 and S. putrefaciens YZ07.

Biofilm and Exopolysaccharide Formation
Biofilms have a strong adhesive ability, and they envelop bacteria, thereby enhancing their resistance to adverse environments [53]. On the surface of food processing equipment, some spoilage microorganisms, and pathogenic microorganisms form biofilms. These biofilms are resistant to disinfectants and are difficult to clear, thus affecting food quality and safety. In this study, both strains of S. putrefaciens produced biofilms; however, YZ-J produced a significantly greater amount of biofilm than YZ08 at both temperatures (4 and 30 • C) tested ( Figure 4B). The genes associated with biofilm formation in YZ08 and YZ-J are listed in Table 2. The key factors regulating biofilm formation of Escherichia coli and Pseudomonas aeruginosa include c-di-GMP regulatory system, the cAMP/Vfr pathway, and the two-component regulatory system GacS-GacA and EnvZ-ompR [54,55]. The presence of the above genes in the genomes of both YZ08 and YZ-J suggested that there may be multiple pathways regulating biofilm formation in two strains. The mechanisms involved in biofilm regulation in Shewanella spp. are poorly understood but are thought to be primarily related to the c-di-GMP pathway. c-di-GMP is synthesized by diguanylate cyclase (DGC) from two molecules of GTP and is decomposed into two molecules of GTP through the activity of phosphodiesterase (PDE) [56]. Several genes encoding DGC and PDE were found in the genomes of both YZ08 and YZ-J (data not shown). However, the cdgC gene encoding c-di-GMP PDE was only found in YZ08 (Table 2). Both Shewanella putrefaciens CN32 and Shewanella oneidensis MR-1 possess a conserved operon containing seven genes [57,58], and this operon also exists in YZ08 and YZ-J. The operon encodes an adhesion protein BpfA; a type I secretion system responsible for the secretion of BpfA into the extracellular compartment (a type I secretion system permease/ATPase, a HlyD family type I secretion periplasmic adaptor subunit, a TolC family outer membrane protein and an OmpA family protein); the protease that regulates BpfA activity (transglutaminase-like cysteine peptidase) and the c-di-GMP receptor protein (EAL domain-containing protein). The secretion of the adhesion protein BpfA in Shewanella promotes bacterial adhesion to solid surface, and the bacteria lacking this protein cannot form biofilms [59].
When the intracellular c-di-GMP content is low, the transcription factor FlrA can promote flagellar operon transcription and repress bpf A operon transcription by directly binding to the promoter region of bpf A, and ultimately biofilm formation is inhibited. When the intracellular c-di-GMP level is high, c-di-GMP binds to and forms a complex with the transcription factor FlrA, thereby relieving the transcriptional activation of flagellar-related genes and the transcriptional repression of the bpf A operon. Eventually, the bacterium undergoes irreversible initiation of adsorption and biofilm formation [59]. c-di-GMP also activates the transcriptional regulator RpoS, thereby upregulating the expression of biofilmassociated genes [24]. The amount of biofilm of YZ-J was greater than that of YZ08, which may be due to the higher content of c-di-GMP and the weak motility in YZ-J. Although there are many regulatory mechanisms for biofilm formation, the mechanism for biofilm formation in Shewanella spp. mainly involves regulation of the secretion of the adhesion protein BpfA by the FlrA factor. Accordingly, we explored the differences in the expression levels of flrA and bpf A between the two strains. The RT-qPCR results showed that the expression of flrA, encoding an inhibitor of biofilm formation, was significantly higher, and that of bpf A significantly lower, in the YZ08 strain than in the YZ-J strain (both p < 0.01) ( Figure 5), which was in line with the higher amount of biofilm formation in strain YZ-J relative to that in strain YZ08.
Exopolysaccharide is an important component of bacterial biofilms, and bacteria can promote microcolony formation and biofilm maturation by regulating exopolysaccharide synthesis [60]. Similar to the pattern of biofilm formation, the levels of exopolysaccharide produced by YZ-J were significantly higher than those generated by YZ08 at the end of storage ( Figure 7E). The genes responsible for exopolysaccharide synthesis in both strains are listed in Table 2. No genes responsible for the biosynthesis of the polysaccharides alginate, Psl, Pel, or that of any other exopolysaccharide, were identified in the genome of either strain. Only glycogen synthesis genes were found. Glucose 6-phosphate is converted to glucose 1-phosphate by the phosphoglucomutase (encoded by pgm), following which glucose 1-phosphate is converted to ADP-glucose through the activity of glucose-1-phosphate adenyltransferase (encoded by glgC). ADP-glucose is subsequently used to extend the α-1,4-glucosidic chain through glycogen synthase (encoded by glgA), after which branching enzyme (encoded by glgB) catalyzes the formation of α-1,6-linked branch chains, yielding glycogen. Glycogen is broken down into glucose by glycogen phosphatase (encoded by glgP) [61]. In our study, RT-qPCR results ( Figure 5) showed that the expression of glgA in S. putrefaciens YZ-J was significantly higher than in S. putrefaciens YZ08 (p < 0.001), which could explain the higher production of exopolysaccharides in the former.

Protease and Lipase
Proteases and lipases secreted by spoilage bacteria hydrolyze, respectively, protein and fat in seafood, thus reducing its quality [62]. The protease and lipase activity of S. putrefaciens YZ08 and YZ-J is shown in Figures 7F and 8. The protease activity of YZ08 was found to be significantly greater than that of YZ-J (p < 0.05). The results also showed that YZ08 had substantially larger protease hydrolysis halos than YZ-J, and that no protease hydrolysis halo was seen for YZ-J at 4 • C ( Figure 8A,B). However, the lipolytic activity of YZ08 was slightly lower than that of YZ-J, although the difference was not significant ( Figure 8E). Exopolysaccharide is an important component of bacterial biofilms, and bacteria can promote microcolony formation and biofilm maturation by regulating exopolysaccharide synthesis [60]. Similar to the pattern of biofilm formation, the levels of exopolysaccharide produced by YZ-J were significantly higher than those generated by YZ08 at the end of storage ( Figure 7E). The genes responsible for exopolysaccharide synthesis in both strains are listed in Table 2. No genes responsible for the biosynthesis of the polysaccharides alginate, Psl, Pel, or that of any other exopolysaccharide, were identified in the genome of either strain. Only glycogen synthesis genes were found. Glucose 6-phosphate is converted to glucose 1-phosphate by the phosphoglucomutase (encoded by pgm), following which glucose 1-phosphate is converted to ADP-glucose through the activity of glucose-1-phosphate adenyltransferase (encoded by glgC). ADP-glucose is subsequently used to extend the α-1,4-glucosidic chain through glycogen synthase (encoded by glgA), after which branching enzyme (encoded by glgB) catalyzes the formation of α-1,6-linked branch chains, yielding glycogen. Glycogen is broken down into glucose by glycogen phosphatase (encoded by glgP) [61]. In our study, RT-qPCR results ( Figure 5) showed that the expression of glgA in S. putrefaciens YZ-J was significantly higher than in S. putrefaciens YZ08 (p < 0.001), which could explain the higher production of exopolysaccharides in the former.

Protease and Lipase
Proteases and lipases secreted by spoilage bacteria hydrolyze, respectively, protein and fat in seafood, thus reducing its quality [62]. The protease and lipase activity of S. putrefaciens YZ08 and YZ-J is shown in Figures 7F and 8. The protease activity of YZ08 was found to be significantly greater than that of YZ-J (p < 0.05). The results also showed that YZ08 had substantially larger protease hydrolysis halos than YZ-J, and that no protease hydrolysis halo was seen for YZ-J at 4 °C ( Figure 8A,B). However, the lipolytic activity of YZ08 was slightly lower than that of YZ-J, although the difference was not significant ( Figure 8E). Genes encoding protease and lipase from in the YZ08 and YZ-J genomes are listed in Table 3. There were differences in the genes encoding proteases that contain signal peptides between YZ08 and YZ-J. Signal peptides in enzymes are necessary for enzyme secretion [63], and extracellular protease secreted by bacteria generally contains a signal peptide. Here, we found that YZ08 contained two genes encoding M48 family metalloproteases (K2227_09265 and K2227_17060) and one encoding an M4 family metallopeptidase (Hap) while YZ-J had only one gene encoding M48 family metalloprotease Genes encoding protease and lipase from in the YZ08 and YZ-J genomes are listed in Table 3. There were differences in the genes encoding proteases that contain signal peptides between YZ08 and YZ-J. Signal peptides in enzymes are necessary for enzyme secretion [63], and extracellular protease secreted by bacteria generally contains a signal peptide. Here, we found that YZ08 contained two genes encoding M48 family metalloproteases (K2227_09265 and K2227_17060) and one encoding an M4 family metallopeptidase (Hap) while YZ-J had only one gene encoding M48 family metalloprotease (K3G22_08175). YZ08, but not YZ-J, also contained a gene encoding an alkaline serine protease. Moreover, we found that YZ-J lacked protease activity at 4 • C (no halo was produced on skimmed milk-containing), which may be related to absence of any gene encoding an alkaline serine protease in this strain, which usually still exhibited activity over a large temperature range (0-50 • C) [64]. YZ08 and YZ-J shared the same lipase encoding gene, likely explaining why the two strains showed similar lipolytic activity. Genes encoding alkaline serine proteases have also been found in S. baltica 128 and S. putrefaciens XY07, while the hap gene was found in only S. baltica 128. Genes encoding lipases were found in both S. baltica 128 and S. putrefaciens XY07. Table 3. Genes encoding proteases and lipases of S. putrefaciens YZ08 and YZ-J.

Gene
Encoded Protein Locus Tag Signal Peptide YZ08 YZ-J spoilage-related metabolism existed between the two strains. Strain YZ08 displayed better growth than YZ-J under NaCl stress, which may be relevant to the presence of more genes encoding sodium:proton antiporter and the high expression of a gene encoding a choline/glycine/proline betaine transporter protein in the YZ08 strain. YZ08 also was found to have greater swimming motility than YZ-J, which was consistent with the greater number of cheX genes found in the former strain. The strong swimming motility and the low transcript levels of the bpf A gene, possibly due to low c-di-GMP content, likely resulted in a low biofilm-forming capacity for YZ08. The lower production of exopolysaccharides in YZ08 relative to YZ-J may be related to the low expression of glgA, which encodes glycogen synthase. The lack of the TMA metabolism-related operon torECADSTR may explain the lower TMA generation in YZ-J. The presence of genes encoding extracellular proteases (alkaline serine protease and M4 family metallopeptidase) may be important factors causing low extracellular protease activity of YZ-J. Overall, some differences in the genetic factors of two strains were consistent with the phenotypic differences. This study contributes to the understanding of the molecular mechanisms underlying the spread, motility, and spoilage activity of two strains of S. putrefaciens.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods11091261/s1, Table S1: quantitative real-time PCR primers used in this study; Table S2: unique genes of S. putrefaciens YZ08 by pan-genome analysis between S. putrefaciens YZ08 and YZ-J.; Table S3: unique genes of S. putrefaciens YZ-J by pan-genome analysis between S. putrefaciens YZ08 and YZ-J.
Author Contributions: Z.Y.: conceptualization, methodology, software, investigation, writing. J.X.: validation, formal analysis, writing-review and editing, examination, funding acquisition. All authors have read and agreed to the published version of the manuscript.